During heart failure, cardiac fibroblasts undergo pathphysiologic changes in cellular phenotype which recapitulate the processes of injury, inflammation, and wound healing. In the failing heart, these responses culminate in cardiac fibrosis, which contributes importantly to the deterioration of myocardial performance. These phenotypic changes include altered proliferation, migration, extracellular matrix synthesis, and production of autocrine-paracrine mediators. Alterations of cardiac fibroblast phenotype are likely driven by humoral substances acting on cell surface receptors to regulate intracellular signaling cascades, leading to activation of defined transcription factors. However, understanding of fibroblast biological responses in terms of coordinated programs of gene transcription is currently limited. The overall hypothesis of this work is that the phenotype of the activated cardiac fibroblast is determined by the combinatorial actions of several key pro-inflammatory cytokines and fibrogenic growth factors. We propose further that this phenotype represents a fundamental change in the transcriptional program of the cell, resulting from interactions of the signaling cascade(s) and downstream nuclear regulators initiated by these agonists. To elucidate the signaling mechanisms which underlie these phenotypic endpoints, the following specific aims are identified: Specific Aim 1 will determine the signaling mechanisms of cytokine and growth factor regulation of fibroblast proliferation; Specific Aim 2 will investigate cytokine and growth factor regulation of fibroblast migration and cytoskeletal organization; Specific Aim 3 will determine signaling mechanisms of cytokine and growth factor regulation of extracellular matrix metabolism; and Specific Aim 4 will define cytokine regulation of autocrine-paracrine mediators of fibrosis; namely, the Renin-Angiotensin and TGFbeta systems. Our experimental system utilizes cultures of cardiac non-myocytes (fibroblasts) from neonatal and adult rats. Investigations will focus on the pro-inflammatory cytokines IL-1, TNFalpha, IL-6, and IFNgamma; and the fibrogenic growth factors Angiotensin II, Aldosterone, and TGFb. Activation of prototypic signaling cascades and their downstream transcription factors, including MAP Kinase-AP-1, NF-kappaB, and JAK-STAT, will be determined. Signaling pathways will be manipulated experimentally using pharmacological inhibitors, and by expression of recombinant signaling molecules using adenovirus vectors. Insights into convergent mechanisms regulating fibroblast phenotype will offer novel strategies for therapeutic management of heart failure.